Photoreduction of reducible materials



United States Patent 2,734,021 PHOTOREDUCTION or nnnucm n MATERIALS -Walter J. Nickerson, New Brunswick, and Joseph R.

" Merkel, Princeton, N. J., assignors to Rutgers Research :and:Endowment .Foundatiom'New Brunswick, N. 3., a nonprofit corporation of N ewJ ersey NoDravving. Application September 17, 1954,

' S erialNo.'456,882

,Qur present invention relates to photoreduction of niaterialsgand"particularly to a method for con- ,visible"light,ivhich maybe either Sunlight or artiigiit}intoienergfl which is usable, for example, in the reduction of reducibleiF hemical compounds. "While'it'is ,recognized that various ways'have been suggested heretofore for converting light energy into the Lrormspr energy, the present invention seeks to accomplish this desirable end by providing a water solu- 'onof eertain chemical materials, which are readily ble,and' whi ch;are sufficiently sensitive to light in visible'range to give a desirable and usable amount of nergyin some other form. For example, it may be :"dtojrediiceson ie chemical material which is of a b Amongfsuch reducibleimaterials are, ver compounds, which are reducible to d 4 whichhave been used for many in thelphotographic'industry. The present inven- PiQYidesia novelianddesirable combination for use with theireduction of these silver com- As a fu rtherexample, a considerable number of dyes and other eolored chemical materials may exist in two states? '(ajan oxidizedjform; and (b) a reducedform.

in such materials havediifereht colors in the oxidized d f c'ir ms respectively. When. the oxidized rmjof one of these materials is reduced in accordance the present invention, 'a'difierent material is produced thereby which iscapableof acting as an indicator in Jae tigdhpreseiib'ebf certain oxidizing agents and com mations thereof 'which is of relatively great Tserisitivityin making "such detections. For this reason thefediicedformbf' 'thes e'ma terials are valuable commercialcommodities which may be produced'from the oxi- 'dizcd foims ifespectiv'ely in accordance with the method the""p'reselnit "invention. Particular examples of this iffypeofns of the invention will appear hereinafter. use invention may be summarized as comprising an u e l sfgiiqe lxe 'i e having t o m .end ldwenalkyLgroups'. In practice, this isoalloxazine is presentrlin 'ithe solution in a' concentration from about lilitfitnllolar up to thei maximum water-soluble concentravol.

tion thereof, i. e., saturation. There is also preferably present in the solution"ametabconiplexing'agent, which m y t k th w ormv of a seque terin as io h s agentL' This material is" used in accordance the h e n w believdto be correc di el n a mp b ga mpo fou nyl teta it h i P esen usually in trace amounts and asanimpu'rit'y in I a 1 e o he n r dien .Q s lu i .s; this metallfrom acti'rig as'ian inhibitdr a' photochemical action "of the iso lo aforesaid.

It'has been found, for

p. 9 h i os c mm cialjm'aterials, such as the commercially available f rrns of the isoalloxaziiieslaccor mg totheip u n ibrQfl i ifi (V ia TB 1 WP 4. other reducible materials which may he used em as aforesaidfand even in e of thewater itse y be se i ma d e P fiolfl' amounts of some metals,.,for example, a1 um, eal iu n, magnesium and zinc; as well iron, copper, chro smaller amounts) are present. It 7 a en n y e ste i hee a trace 'coricentrationswill actj to characteristics of th lox everyone eiinie egsui introduced'in the s'olut10n,f th v e ls, n f p as afor e said, will bebouud' u insome n ietal m l the w n tur o t hi an tn ,7 inwhich the 'riitfal or, intals no longer retainthen haracte'rfas inhibitors. The' us'e of a me a ecomp Xni'ga m is preferably, therefore, 1a permeate present'inve The p'r'ese'nt' invention grew outof'certain-invesftig which aredescribed inapubn atipnpr'tne pr'e ventors, eminen e. Light Activation Phenomenon Enzymatic and Nonenzymatic Reductionof 'rj prazemm sans, published in vol. '39, No.9, pp. 901-905; Proceedi gs f theNat O lal Academy of S iences, s temper 1953. The investigation was carriedonfurtherand a subsequent article also writtenbydhe present inventors 'andlentitled Riboflavin as alPhotocat alyst and Hydrogen Carrier in Photochemical Reduction, waspublished in 14 (1954) Biochimica' etfBiophysica 303 -311.

' Turning now to the essential active ingredientswof, the combination and the which they play, it was-roan thatone such essential ingredient is riboflavin a equivalents, which may be generically described'as isoalloxazine having the formula" and. possible "substituents as above set forth." The variations hereinab gested for this ingredient comprise all'the" presently equivalents thereof. Other materials have been triedl'b ut have proven undesirable for reasons which willpreseht'ly appear. It will be understood that each',' material. tested was dissolved in distilled water; A m ta -complain agent in a kind and amount found to be operativein conjunction with'certain of the preferred materials ashereim above set forth was added and the solution was subjected to light radiation of, a type and under conditions lwhich were found. to givev the best resultsFusing riboflavin the fphosphate derivativesthereof, both ,of whiehwere fully operative as herein noted. Unless otherwise. noted, eachmaterial was. te ted, as to its photochemical action in reducing a dye .and ,also its. ability to give a measurable electrical potential upon potentiometric test. materials which were so'tested include: n

(1') Uranyl salts (uranyl acetate and uranyl sulfate). These materials were ineffective either to reduce dyes "or in solution'as the'electrolyte in a cell." (2) Quinone-lhydroquinone mixture.

to give any substantially electrical'response used Again negative Siich' other arsaoar. t

results both for dye reduction and potentiometrically were found.

(3) Trypaflavin (3, 6, diamino-lO-methyl acridinium chloride). This material was effective in a slight degree only in reducing a standard dye, triphenyltetrazolium chloride (herein abbreviated as TTC).

(4) Acriflavin. The results were the same as those described above in connection with (3) trypafiavin.

(5) Acridine orange. This material was found to be inactive both from the point of view of dye reduction and potentiometrically.

(6) Quinoline yellow. This material was found to be inactive both from the point of view of dye reduction and potentiometrically.

(7) Vitamin A. This material seems to be operative to a. certain limited extent only as a substitute for riboflavin, in that it is somewhat light-sensitive, but the response thereof to light is much slower than in the case of riboflavin, and further it is very slightly soluble in water, so that it is not considered to be within the purview of the present invention.

(8) Chlortetracycline. This material was found to have no apparent action in reducing a dye (TTC).

(9) Quinine. This material was found to give no potentiometric response. It was not tested with a dye; but in general every material which was found to be capable of reducing a dye was found also to give a potentiometric response when so tested.

(10) Atabrine. This material was found not only to be useless as a substitute for riboflavin, but even acted as an inhibitor when added to a solution containing riboflavin.

(11) Acridine. This material developed a small potential when used in a cell in lieu of riboflavin or its equivalents as herein presented, but the results were so small that it is not considered as an equivalent for the purposes of the present application and is not to be considered as coming within the purview of the present invention.

The present invention as aforesaid is an outgrowth of certain work reported in the article published in the Proceedings of the National Academy of Sciences and hereinabove referred to, in which we reported on certain studies on the enzymatic reduction of tetrazolium salts by non-proliferating suspensions of yeast and wherein it was noted that light had a very substantial eifect upon the rate of reduction of the dye. It was further noted at that time that riboflavin, one of the isoalloxazines hereinabove referred to, clearly stimulated the photo reduction of the dye. This led us to a further detailed study, primarily from a purely chemical point of view, to determine the essential characteristics of the photoreduction reaction noted as aforesaid. The results of certain of these studies are reported in our second paper published in Biochimica et Biophysica Acta and also hereinabove referred to.

We have found that riboflavin, as an example of isoalloxazines, apparently acts as a photocatalyst, in that it appears to be activated by visible light, so as to catalyze the reduction of a reducible material which may be in solution with it.

Our present theory is that the light-activated riboflavin probably splits water, acting as a hydrogen carrier to convey the hydrogen from the water to a reducible material. This action, however, i. e., the eifective transfer of hydrogen to a reducible material, appears to be inhibited to a major extent by the presence of very minute amounts of metals, such as are'introduced into a solution of riboflavin, possibly as impurities in the commercially available riboflavin itself, possibly as impurities present in the water and/ or possibly as impurities in the reducible material introduced into the system. In any event, it is known that usually (with an exception hereinafter set forth) in the absence of a metal-complexing agent, which may be a metal chelator and may also be classed as a sequestering agent, the reducing action is substantially prevented. In the presence of such an agent, capable of tying up minute amounts of metal introduced as aforesaid into some metal-organic complex, the inhibitory action of the metal or metals ceases and the photo-sensitive action of riboflavin, which we have discovered to exist and which may be attributed to the theories hereinabove given, does in fact take place.

There are many known metal chelators with which the art is quite familiar. There are also many materials which while not strictly chelators, may be classed under the broader term of metal-complexing agents. Any such material, which will be effective to tie the metal present up in a complex such that it will not inhibit the photoreduction of riboflavin and its equivalents, is to be considered to be within the purview of the present invention. Among such materials which have been tried and found to be fully and completely operative are the following:

(a) Ethyleneaminetetraacetic acid and the watersoluble salts thereof. The disodium salt of this acid is commercially available from several commercial sources. This material is sometimes abbreviated NazEDTA.

(b) Cysteine. This material appears to operate to obtain desirable results at pH 7 and above.

(c) Mercaptosuccinic acid. This material seems to be operative throughout the entire pH range as hereinafter noted.

(a?) Carboxymethylmercaptosuccinic acid. This ma.- terial also appears to be operative throughout the entire pH range hereinafter noted.

The following materials were tried as metal-complexing agents, but operated either so poorly that their use was believed relatively undesirable or were completely inoperative as compared with some of the desirable materials above noted: sodium citrate, sodium oxalate, glycine (at pH 8), potassium cyanide, dithiozone, various water-soluble salts of phosphoric acid, 8-hydroxyquinoline, o-phenanthroline, Actinomycin and glutathione.

As a further portion of the present studies, tests were made as above generally referred to, to determine the materials which could be substituted for the riboflavin, originally tried, as this ingredient of the composition according to the present invention. The materials which are inoperative for this purpose have been outlined above. The materials which are operative have also been outlined above. Each of these operative materials has in fact been tested as hereinafter noted, and found to be eifective either in the photo-reduction of some reducible material or in effecting a conversion of visible light energy to electrical energy as determined by potentiometric measurement, or both.

The concentration of the isoalloxazine material as aforesaid which is required to produce desirable results has been found to be from about 10- molar up to saturation. The low limit of concentration is not narrowly critical. It has been found, for example, that the effective light sensitivity as measured by the potentiometer in a solution gives a progressively less response as the concentration is reduced, considering a fixed light intensity and time of light exposure or radiation. As the com centration is reduced to about 10- molar, for example, a three seconds radiation with a 375 watt incandescent bulb at a distance of about 15 cm., which sufliced with more concentrated solutions of riboflavin to give substantial and measurable results, becomes ineifective to give similar measurable results. On the other hand, with the same intensity-distance illumination, but with a longer time, some such results could be obtained even with this concentration. It is considered, however, that this is about the practical lower limit of concentration which is practically useful in accordance with the invention.

The maximum concentration in the case of many materials, such as riboflavin, is chosen as the saturation concentration at the temperature. which is used, i. e. room temperature for example. This is due to the fact that riboflavin per .se is, of course, relatively slightlywsolulgle vi Ir. With: other materials coming within the ge'neral finition hereinabove given of an isoalloxazine, the -saturation concentration is substantially higher and vneed not ,be approached even for maximum results. Ithas been found in this connection that maximum results are attained at concentrations substantially less thantsaturan a tho h on en r t n P to a r o a s i operative to give desirable results. The optimum concentrati on;for eachmaterial is a matter which can be easily eterminedbythoseskilled inthe art from the foregoing. or example, .it has been found with riboflavin that c9 1sent:atiq mo is a s s l a 'sfle tive concentration for practical use. This sarnegoncentionis alsoretlective even with some of the moresoluble nihersof the generalclassification of isoalloxazines given above. On the other hand, in theevent .that'lt'he purpose as herein noted is to reduce areducible material, Y,- e, concen tration of the isoalloxazine material may be 'creasediwithin the solubility range if ,a relatively enarnount of reducible. material is to be reduced in I The rconcqntrfition .of the vmetal-complexing ,agent is 5 el a matter ofexperience with a particular combinan o h Im f 1 be n ed 1 1 0 F o fi theoretical point of .yiew and pursuant to our present t heories, it isnecessarythat there be asufificientamount o the tnetal-cornpl exing agent to react with all the metal lpresentQfrom whatever source it may come, in other words, the stoichiometric amount. In view ,of the fa ct at the metal or metalsin questionarepresent.byinadence rather than by design, it is. practically imposbl to calculate the fstoichiornetric amount. As aireif i usu l y th pra ti to h v pre en alon ZIargerarn untofthemetal-cornplexingagent than a uid be re so l s rsst df b v lss 4 g meta Presen f e am l t ha bee ssn a agconcentration of NasEDTA (as definedabove), of -molar iseifective in a system containing riboflavl u at afioncentration of 4 l0- molarand distilled water along with a cencentration-of 1 0 molar triphenyltetrazolium chloride (TTC). If, however, the solution is made up using tap water in lieu of distilled water, it is reasonably expected andhas been foundthat a somewhat higher'concentration of the metal-complexing agent .is. desirable.

Ithas-been found that aqueous solutions according to .the presentinvention are fully operative at pl-l values from about 5 t o-a bo ut ,9, n either ofthese limits being particularly narrowly critical. Under ordinary circumstances, the ingredient materials along with relatively pure (and neutral) water will -give solutions within this range. "It may be, however,-in some instances that buflering may be necessary to bring theso lution within general'range, if so any suitable "and conventional butie ring agents may be used.

The reason for choosing these pH limits are as follows: As to the lower limit, it is found that photoreduction reactions become so slow at the more highly acid end of the pH range given above that it is not practical to operate with a pH lower than about 5. This limit may, therefore, be chosen more or less arbitrarily as a practical limit for purposes of the present invention. The higher range limit, i. e. the limit in an alkaline direction, is chosen because as this limit is approached or passed, the isoalloxazine element of the combination appears to be destroyed in high alkaline solutions. Thus, from a practical point of view, the invention operates successfully only within about the range given.

The present invention has been tested practically in the reduction of a considerable number of dilferent reducible materials, many of which are dyes, and certain of which have peculiar utility in their reduced conditions. In connection with the reducing capacity of systems according to the present invention, a group of tests were Triphenyltetrazolium chloride lOrange'tetrazolium xlodine 'Methylener-blue Toluidine.:blue :Rosindulin ZB Trypan blue os-Dinitrobenzene .p-Nitrobenzalde'hyde =Mercuric chloride' -.-Janus.'grecn=-:B'

Malachiteigreen Cytochrome c Nile blue Phenol blue 0 Resazurin aPhfiilOl-illdO-Z, 6 .dichlorophenol :Silver salts,.asthe.nitrate.

.spectively. -;-Thus, the.,.present .rnethod may bev utilized for preparing these materials:intheinrespectively reduced statesjp which. state or condition the respectivematerials .may then .be recovered :from the solution due t0 heir 3lessersolubility,.andsold, or otherwiseused as a product .of the Eprocess. ;In many instances the reducedmaiterials [maysbe used as indicators ;in other systems Ito detect .andindicate the presence of oxidizing material. or

materials in such the systems. Th s, the P s ;me thod :igpraetically ,eifective in producing indicators,

{which are of ,greatuse in; biological research tests, such indicators, for example,=being.methyleneblue, to u din blue, rosinduline 12B, trypan :blue, o-dinitrobenzene, .nitrobenzaldehyde, mercuric chloride, janus green -B ,malachitelgreen, cytochromerc, inileblue, phenol .blue,

resazurin andphenol-indo-Z, 16 dichlorophenol.

While ,a considera le; number of' oxi izable an me- .-,ducibie,mater ials;.-have-been listed herein ,andthedesirable properties of the present process has been tested thereagainst, this list is by no means exhaustive, but merely indicates certain materials which have in fact been tested, rather than purporting to indicate all the materials to which the present process is applicable. As a practical matter, the only limitation is that the reducible chemical material, which may be reduced according to the present process or method, must be one having a redox potential which is substantially more positive than that of the isoalloxazine which is used in carrying out the process.

An additional possible use of the present invention, however, remains to be described, that is, in connection with the photoreproduction of material by utilizing the capacity of the system for photoreduction of a reducible material, for example, tetrazolium compounds, so as selectively to reduce such salts at any portion only of the system which is directly subjected to light radiation. If the system is created in a slightly moist or semi-solid bodiments of the invention, it has been found by actual tests that portions only of such a semi-solid film including the several ingredients hereinabove described and further including a reducible tetrazolium salt may be exposed to light in a manner similar to the light-exposure of a photov graphic negative, since it has been found that the system is effective in reducing other materials, such as silver compounds. The portion so exposed to light will be found to have reduced tetrazolium compounds thereon, while portions not so exposed will not have the tetrazolium salts in a non-reduced form. A photographic reproduction can, therefore, be made by this process. It is contemplated that other photo-reducible materials may be substituted for the tetrazolium compounds actually tried by following the teachings of the present invention and within the purview thereof.

While there is herein described a number of embodiments of the invention, other possible equivalents or embodiments thereof will occur to those skilled in the art from the foregoing description. We do not wish to be limited, therefore, except by the scope of the appended claims, which are to be construed validly as broadly as the state of the art permits.

What is claimed is:

1. The method of efliecting the photoreduction of a reducible chemical material, comprising the steps of preparing an aqueous solution of an isoalloxazine having the formula:

wherein X is selected from the group consisting of ribitol (CH (CHOH) -CH OH), phosphate derivatives of ribitol, tetraacetylated ribitol (CH (CH-OOC-CH -CH OOC-CH succinate (OOC-(CH -COOH), and water-soluble salts of succinate, and in which R and R are each selected from the group consisting of hydrogen and lower alkyl groups, said isoalloxazine being present in a concentration from about 10 molar up to the maximum water-soluble concentration thereof, also establishing in said solution as a solute therein a metal-complexing agent in a concentration at least stoichiometrically equivalent to the total of the metals present in the solution which are capable of combining with said metal-complexing agent to form metal-organic complexes, also establishing in said solution as a solute therein a reducible chemical material to be reduced by photoreduction in the solution and in which said reducible chemical material has a redox potential substantially more positive than that of said isoalloxazine which is present in said solution, and exposing the solution made up as aforesaid and including said reducible material to visible light to effect photoreduction of said reducible material.

2. T he method in accordance with claim 1, in which said metal-complexing agent is a soluble salt of ethylenediamine-tetra-acetic acid.

3. The method in accordance with claim 1, in which said metal-complexing agent is cysteine and in which the pH of the solution is at least about 7.

4. The method in accordance with claim 1, in which said metal-complexing agent is mercapto-succinic acid.

.5. The method in accordance with claim 1, in which said metal-complexing agent is carboxy-methyl-mercapto succinic acid.

6. The method in accordance with claim 1, in which the substituents, R and R in the isoalloxazine formula given are each methyl.

7. The method in accordance with claim 1, in which said reducible chemical material is one which changes color when reduced and further is one in which the reduced form may be reoxidized to its initial state by bringing into contact therewith an oxidizing chemical agent, whereby the reduced material produced as a prodnet of the present process may be used as an indicator to show the presence of a chemical agent capable of reoxidizing said reduced chemical material.

8. The method in accordance with claim 1, in which said reducible chemical material is a dye, whichhas a substantial water-solubility in both its oxidized and re duced states.

9. The method in accordance with claim 1, in which said reducible chemical material is one having a re- References Cited in the file of this patent UNITED STATES PATENTS 1,887,531 Wein Nov. 15, 1932 1,941,494 Ruben Ian. 2, 1934 2,604,442 Lambert et al July 22, 1952 OTHER REFERENCES Proceedings of the National Academy of Sciences, vol. 39, No. 9 (Sept. 1953), pp. 901-905.

Biochemica et Biophysica Acta, vol. 14 (1954), pp. 393-311.

Oster: Photographic Engineering, vol. (1953), pp- 173-173.

. l tout-mm. 

1. THE METHOD OF EFFECTING THE PHOTOREDUCTION OF A REDUCIBLE CHEMICAL MATERIAL, COMPRISING THE STEPS OF PREPARING AN AQUEOUS SOLUTION OF AN ISOALLOXAZINE HAVING THE FORMULA: 